A sensitive LC-MS/MS method for the determination of free maytansinoid DM4 concentrations - Method development, validation, and application to the nonclinical studies of antitumor agent DM4 conjugated hu-anti-Cripto MAb B3F6 (B3F6-DM4) in rats and monkeys

摘要

A sensitive, specific, and high throughput method was developed and validated for the quantitation of free maytansinoid DM4 in cynomolgus monkey and Sprague-Dawley rat plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The Sciex API 5000 mass spectrometer was operated in multiple-reaction monitoring (MRM) and electrospray positive ionization mode. An MS/MS transition of 780 → 216 was used to monitor the analyte DM4 and 635 → 547 for the internal standard (IS) ansamitocin P-3. A liquid-liquid extraction was utilized for sample pre-treatment with a volume of 100 μL plasma. A Thermo Hypersil Gold PFP column was used for chromatographic separation with a 2.0 min HPLC gradient. The quantitation range of the method was 0.500-100 ng/mL with a lower limit of quantitation of 0.500 ng/mL. The method was validated in monkey and rat plasma according to FDA guidance on Bioanalytical Method Validation (2001). The intra- and inter-day precision and accuracy were found to be within the FDA recommended acceptance criteria. The validated method was employed to monitor the free DM4 levels in plasma in the IND-enabling toxicology studies of antitumor agent DM4 conjugated hu-anti-Cripto MAb B3F6 (B3F6-DM4).